The goal of the principal investigator is to continue to develop intellectual, technical, and analytical skills to become an independently funded physician-scientist investigator in microbial pathogenesis, examining the effect of bacterial products on the immune system. The program to achieve this goal will consist of additional didactic and laboratory training in basic immunology, lymphocyte signal transduction, and microbial and molecular genetics. Bacterial immunomodulatory products are of utmost importance in infectious diseases and their prevention. For example, C. difficile toxin A and B are implicated in the development of pseudomembranous enterocolitis, and cholera toxin functions as an adjuvant in oral immunization. In this proposal we will continue to characterize a novel toxin from Enteropathogenic E. coli (EPEC), resulting in marked inhibition of T cell activation. The inhibitory gene, lifA (lymphocyte inhibitory activity), encodes for a protein with the putative size of 366kDa. The lifA gene product, lymphostatin, bears significant similarity to the N-terminus of large Clostridial cytotoxins, encoding for a glucosyltransferase motif, which is critical for their specific activity. Similar immunosuppressive genes and biological activity have been identified in related bacteria, including other EPEC strains, Enterohemorrhagic E. coli, and the mouse pathogen C. rodentium. Our hypothesis is that the glucosyltransferase motif in lymphostatin is critical for the observed immunosuppression, leading to inhibition of defined lymphocyte subpopulations of the adaptive immune response and allowing firm establishment of enteric Gram negative infection. To test the hypothesis, we propose: Aim 1: To identify the co-substrate and target molecule(s) in lymphocytes exposed to lymphostatin. Aim 2: To investigate intracellular activation pathways in defined lymphocyte populations affected by lymphostatin, resulting in suppression of IL-2, IL-4, and IFN-gamma expression. Aim 3: To investigate whether lymphostatin suppresses the mucosal adaptive immune response and firmly establishes C. rodentium enteric infection in vivo. The proposed research project will contribute to the understanding of immune mechanisms involved in the pathogenesis of chronic infectious diarrhea and gastrointestinal inflammation as seen in Crohn's disease and ulcerative colitis.